Effect of the Ito activator NS5806 on cloned Kv4 channels depends on the accessory protein KChIP2

BACKGROUND AND PURPOSE

The compound NS5806 increases the transient outward current (I_to) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, I_to is thought to be mediated by K_V4.3 and various ancillary proteins, yet, the exact subunit composition of I_to channels is still debated. Here we characterize the effect of NS5806 on heterologously expressed putative I_to channel subunits and other potassium channels.

EXPERIMENTAL APPROACH

Cloned K_V4 channels were co-expressed with KChIP2, DPP6, DPP10, KCNE2, KCNE3 and K_V1.4 in Xenopus laevis oocytes or CHO-K1 cells.

KEY RESULTS

NS5806 increased K_V4.3/KChIP2 peak current amplitudes with an EC₅₀ of 5.3 ± 1.5µM and significantly slowed current decay. KCNE2, KCNE3, DPP6 and DPP10 modulated K_V4.3 currents and the response to NS5806, but current decay was slowed only in complexes containing KChIP2. The effect of NS5806 on K_V4.2 was similar to that on K_V4.3, and current decay was only slowed in presence of KChIP2. However, for K_V4.1, the slowing of current decay by NS5806 was independent of KChIP2. K_V1.4 was strongly inhibited by 10 µM NS5806 and K_V1.5 was inhibited to a smaller extent. Effects of NS5806 on kinetics of currents generated by K_V4.3/KChIP2/DPP6 with K_V1.4 in oocytes could reproduce those on cardiac I_to in canine ventricular myocytes. K_V7.1, K_V11.1 and K_ir2 currents were unaffected by NS5806.

CONCLUSION AND IMPLICATIONS

NS5806 modulated K_V4 channel gating depending on the presence of KChIP2, suggesting that NS5806 can potentially be used to address the molecular composition as well as the physiological role of cardiac I_to.     

The transmembrane β-subunits KCNE1, KCNE2, and DPP6 modify pharmacological effects of the antiarrhythmic agent tedisamil on the transient outward current I to

Accessory β-subunits modulate the pharmacology of ion channel blockers. The aim was to investigate differences in effects of the antiarrhythmic agent and open-channel blocker tedisamil on transient outward current I _to (Kv4.3) when coexpressed with β-subunits potassium voltage-gated channel, Isk-related family, member 1 (KCNE1), potassium voltage-gated channel, Isk-related family, member 2 (KCNE2), or dipeptidyl-aminopeptidase-like protein 6 (DPP6) which modulate I _to kinetics. Tedisamil inhibited I _to with IC₅₀ values of 16 μM for Kv4.3+KChIP2, 11 μM in the presence of KCNE1, and 14 μM for KCNE2. Values were higher in the presence of DPP6 or DPP6+KCNE2 (35 and 26 μM). K _d values of tedisamil binding and rate constants were not affected by KCNE or DPP6. I _to kinetics were accelerated by KCNE and DPP6, inactivation to a larger extent with DPP6. Tedisamil did not affect activation time course but apparently accelerated inactivation in all channel subunit combinations tested. Deletion of the intracellular domain of KCNE2 or DPP6 resulted in slowing of kinetics and increased tedisamil sensitivity (IC₅₀ 4 and 7 μM). It is concluded that apparent effects of DPP6 and deletion mutants (KCNE2 and DPP6) are due to the acceleration or slowing effects of the β-subunits on I _to kinetics.     

Dihydropyridine Ca2+ channel antagonists and agonists block Kv4.2, Kv4.3 and Kv1.4 K+ channels expressed in HEK293 cells

(1) We have determined the molecular basis of nicardipine-induced block of cardiac transient outward K(+) currents (I(to)). Inhibition of I(to) was studied using cloned voltage-dependent K(+) channels (Kv) channels, rat Kv4.3L, Kv4.2, and Kv1.4, expressed in human embryonic kidney cell line 293 (HEK293) cells. (2) Application of the dihydropyridine Ca(2+) channel antagonist, nicardipine, accelerated the inactivation rate and reduced the peak amplitude of Kv4.3L currents in a concentration-dependent manner (IC(50): 0.42 micro M). The dihydropyridine (DHP) Ca(2+) channel agonist, Bay K 8644, also blocked this K(+) current (IC(50): 1.74 micro M). (3) Nicardipine (1 micro M) slightly, but significantly, shifted the voltage dependence of activation and steady-state inactivation to more negative potentials, and also slowed markedly the recovery from inactivation of Kv4.3L currents. (4) Coexpression of K(+) channel-interacting protein 2 (KChIP2) significantly slowed the inactivation of Kv4.3L currents as expected. However, the features of DHP-induced block of K(+) current were not substantially altered. (5) Nicardipine exhibited similar block of Kv1.4 and Kv4.2 channels stably expressed in HEK293 cells; IC(50)'s were 0.80 and 0.62 micro M, respectively. (6) Thus, at submicromolar concentrations, DHP Ca(2+) antagonist and agonist inhibit Kv4.3L and have similar inhibiting effects on other components of cardiac I(to), Kv4.2 and Kv1.4.     

Effects of flecainide and quinidine on Kv4.2 currents: voltage dependence and role of S6 valines

1. The effects of flecainide and quinidine were studied on wild-type Kv4.2 channels (Kv4.2WT), channels with deletion of the N-terminal domain (N-del) and channels with mutations in the valine residues located at positions 402 and 404 in the presence (V[402,404]I) or in the absence (N-del/V[402,404]I) of the N-terminus. 2. The experiments were performed at 37 degrees C on COS7 cells using the whole-cell configuration of the patch-clamp technique. 3. Flecainide and quinidine inhibited Kv4.2WT currents in a concentration-dependent manner (IC(50)=23.6+/-1.1 and 12.0+/-1.4 microMat +50 mV, respectively), similar to their potency for the rest of the constructs at the same voltage. In Kv4.2WT channels, flecainide- and quinidine-induced block increased as channel inactivation increased. In addition, the inhibition produced by quinidine, but not by flecainide, increased significantly at positive test potentials. Similar effects were observed in N-del channels. However, in V[402,404]I and N-del/V[402,404]I channels, the voltage dependence of block by both quinidine and flecainide was lost, without significant modifications in potency at +50 mV. 4. These results point to an important role for S6 valines at positions 402 and 404 in mediating voltage-dependent block by quinidine and flecainide.     

Comparison of the effects of antiarrhythmic drugs flecainide and verapamil on fKv1.4ΔN channel currents in Xenopus oocytes

Aim:

To study the effects of Na⁺ channel blocker flecainide and L-type Ca²⁺ channel antagonist verapamil on the voltage-gated fKv1.4ΔN channel, an N-terminal-deleted mutant of the ferret Kv1.4 K⁺ channel.

Methods:

fKv1.4ΔN channels were stably expressed in Xenopus oocytes. The K⁺ currents were recorded using a two-electrode voltage-clamp technique. The drugs were administered through superfusion.

Results:

fKv1.4ΔN currents displayed slow inactivation, with a half-inactivation potential of −41.74 mV and a slow recovery from inactivation (τ=1.90 s at −90 mV). Flecainide and verapamil blocked the currents with IC₅₀ values of 512.29±56.92 and 260.71±18.50 μmol/L, respectively. The blocking action of the drugs showed opposite voltage-dependence: it was enhanced with depolarization for flecainide, and was attenuated with depolarization for verapamil. Both the drugs exerted state-dependent blockade on fKv1.4ΔN currents, but verapamil showed a stronger use-dependent blockage compared with flecainide. Flecainide accelerated the C-type inactivation rate without affecting the recovery kinetics and the steady-state activation. Verapamil also accelerated the inactivation kinetics of the currents, but unlike flecainide, it affected both the recovery and the steady-state activation, causing slower recovery of fKv1.4ΔN channel and a depolarizing shift of the steady-state activation curve.

Conclusion:

The results demonstrate that widely used antiarrhythmic drugs flecainide and verapamil substantially inhibit fKv1.4ΔN channels expressed in Xenopus oocytes by binding to the open state of the channels. Therefore, caution should be taken when these drugs are administered in combination with K⁺ channel blockers to treat arrhythmia.     

Effect of mosapride on Kv4.3 potassium channels expressed in CHO cells

Mosapride and cisapride are gastroprokinetic agents with 5-hydroxytryptamine₄ receptor agonist activity and have been widely used in the treatment of a variety of gastrointestinal disorders. The effects of mosapride and cisapride on cloned Kv4.3 channels stably expressed in Chinese hamster ovary cells were investigated using the whole-cell patch-clamp technique. Mosapride and cisapride inhibited Kv4.3 in a concentration-dependent manner with IC₅₀ values of 15.2 and 9.8 μM, respectively. Mosapride accelerated the rate of inactivation and activation of Kv4.3 in a concentration-dependent manner and thereby decreased the time to peak. The rate constants of association (k ₊₁) and dissociation (k _?1) for mosapride were 9.9 μM^?1 s^?1 and 151.3 s^?1, respectively. The K _D (k _?1/k ₊₁) was 16.2 μM, similar to the IC₅₀ value calculated from the concentration–response curve. Voltage-dependent inhibition by mosapride was observed in the voltage range for channel opening but was not observed over a voltage range in which all Kv4.3 channels were open. Both the steady-state activation and inactivation curves of Kv4.3 were shifted in the hyperpolarizing direction in the presence of mosapride. Mosapride also caused a substantial acceleration in closed-state inactivation of Kv4.3. Mosapride produced use-dependent inhibition, which was consistent with a slow recovery from inactivation of Kv4.3. M1 and norcisapride, the major metabolites of mosapride and cisapride, respectively, had little or no effect on Kv4.3. These results indicate that mosapride inhibits Kv4.3 by both preferential binding to the open state of the channels during depolarization and acceleration of the closed-state inactivation at subthreshold potentials.     

Blockade by N-3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells.

1. The Kv4.3 gene is believed to encode a large proportion of the transient outward current (Ito), responsible for the early phase of repolarization of the human cardiac action potential. There is evidence that this current is involved in the dispersion of refractoriness which develops during myocardial ischaemia and which predisposes to the development of potentially fatal ventricular tachyarrhythmias. 2. Epidemiological, clinical, animal, and cellular studies indicate that these arrhythmias may be ameliorated in myocardial ischaemia by n-3 polyunsaturated fatty acids (n-3 PUFA) present in fish oils. 3. We describe stable transfection of the Kv4.3 gene into a mammalian cell line (Chinese hamster ovary cells), and using patch clamp techniques have shown that the resulting current closely resembles human Ito. 4. The current is rapidly activating and inactivating, with both processes being well fit by double exponential functions (time constants of 3.8 +/- 0.2 and 5.3 +/- 0.4 ms for activation and 20.0 +/- 1.2 and 96.6+/-6.7 ms for inactivation at +45 mV at 23 degrees C). Activation and steady state inactivation both show voltage dependence (V1/2 of activation= -6.7+/-2.5 mV, V1,2 of steady state inactivation= -51.3+/-0.2 mV at 23 degrees C). Current inactivation and recovery from inactivation are faster at physiologic temperature (37 degrees C) compared to room temperature (23 degrees C). 5. The n-3 PUFA docosahexaenoic acid blocks the Kv4.3 current with an IC50 of 3.6 micromol L(-1). Blockade of the transient outward current may be an important mechanism by which n-3 PUFA provide protection against the development of ventricular fibrillation during myocardial ischaemia.     

Block of cloned Kv4.3 potassium channels by dapoxetine

Dapoxetine, a short-acting selective serotonin reuptake inhibitor, is widely prescribed for the treatment of patients with premature ejaculation. The effects of dapoxetine were examined on cloned Kv4.3 channels stably expressed in Chinese hamster ovary cells using the whole-cell patch-clamp technique. Dapoxetine not only reduced the peak amplitude of Kv4.3 currents but also accelerated the decay rate of current inactivation in a concentration-dependent manner. Thus, the concentration-dependent reduction in Kv4.3 was measured from the integral of the current during the depolarizing pulse. Dapoxetine decreased the integral of the Kv4.3 currents over the duration of a depolarizing pulse with an IC(50) of 5.3 μM. Analysis of the time dependence of the block gave estimates of an association rate constant (k(+1)) of 3.9 μM(-1)s(-1) and a dissociation rate constant (k(-1)) of 25.6s(-1). The K(D) (k(-1)/k(+1)) was 6.5 μM, similar to the IC(50) value calculated from the concentration-response curve. The block of Kv4.3 by dapoxetine was highly voltage-dependent at a membrane potential coinciding with the activation of the channels. The additional block by dapoxetine displayed a shallow voltage dependence (δ=0.21) in the full activation voltage range. The steady-state inactivation curves were shifted in the hyperpolarizing direction in the presence of dapoxetine. Dapoxetine also caused a substantial acceleration in closed-state inactivation. Dapoxetine produced a significant use-dependent block, which was accompanied by a delayed recovery from inactivation of Kv4.3 currents. These results indicated that dapoxetine potently blocks Kv4.3 currents by both preferentially binding to the open state of the channels and accelerating the closed-state inactivation. These data could provide insight into the mechanism underlying some of the therapeutic actions of this drug.     

Basic arrhythmogenic mechanisms in both inherited and acquired long QT syndrome

The heterologous expression system will provide clues for understanding the basic mechanism of arrhythmogenicity in both inherited and acquired long QT syndrome, which are reviewed here, with emphasis on the K+ channels. Endothelin is implicated in the morphological and electrical remodeling of cardiac muscles in heart failure. The effects of endothelin on the transient outward K+ currents (Ito) were compared between Kv1.4 (rich in endocardial muscle) and Kv4.3 (rich in epicardial muscle) channels in the Xenopus oocytes expression system. Both Itos were decreased by stimulation of endothelin receptor ETA coexpressed with the K+ channels. Ito of Kv1.4 was decreased by about 85% after 10(-8) M ET-1, whereas that of Kv4.3 was decreased by about 60%. By mutagenesis experiments, we identified two phosphorylation sites of PKC and CaMKII in Kv1.4 responsible for the decrease in Ito by ET-1. In Kv4.3 we identified a PKC phosphorylation site that is partly responsible for the decrease. Differences in the suppression of Ito could be due to the differences in intracellular signaling including the number of phosphorylation sites. These findings show some of the molecular mechanisms of ventricular arrhythmias in heart failure, resulting in dispersion and prolongation of action potential which elicit reentry and after depolarization.     

Inhibition of cardiac Kv1.5 and Kv4.3 potassium channels by the class Ia anti-arrhythmic ajmaline: mode of action

Ajmaline is a class Ia anti-arrhythmic compound that is widely used for the diagnosis of Brugada syndrome and the acute treatment of atrial or ventricular tachycardia. For ajmaline, inhibitory effects on a variety of cardiac K⁺ channels have been observed, including cardiac Kv1 and Kv4 channels. However, the exact pharmacological properties of channel blockade have not yet been addressed adequately. Using two different expression systems, we analysed pharmacological effects of ajmaline on the potassium channels Kv1.5 and Kv4.3 underlying cardiac I _Kur and I _to current, respectively. When expressed in a mammalian cell line, we find that ajmaline inhibits Kv1.5 and Kv4.3 with an IC₅₀ of 1.70 and 2.66 μM, respectively. Pharmacological properties were further analysed using the Xenopus expression system. We find that ajmaline is an open channel inhibitor of cardiac Kv1.5 and Kv4.3 channels. Whereas ajmaline results in a mild leftward shift of Kv1.5 activation curve, no significant effect on Kv4.3 channel activation could be observed. Ajmaline did not significantly affect channel inactivation kinetics. Onset of block was fast. For Kv4.3 channels, no significant effect on recovery from inactivation or channel deactivation could be observed. Furthermore, there was no use-dependence of block. Taken together, we show that ajmaline inhibits cardiac Kv1.5 and Kv4.3 channels at therapeutic concentrations. These data add to the current understanding of the electrophysiological basis of anti-arrhythmic action of ajmaline.     

Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts

Aim:

Neferine is an isoquinoline alkaloid isolated from seed embryos of Nelumbo nucifera (Gaertn), which has a variety of biological activities. In this study we examined the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (I_to) in rabbit heart, and on ex vivo electrophysiology of rabbit hearts.

Methods:

Whole-cell Kv4.3 currents were recorded in HEK293 cells expressing human cardiac Kv4.3 channels using patch-clamp technique. Arterially perfused wedges of rabbit left ventricles (LV) were prepared, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG).

Results:

Neferine (0.1–100 μmol/L) dose-dependently and reversibly inhibited Kv4.3 currents (the IC₅₀ value was 8.437 μmol/L, and the maximal inhibition at 100 μmol/L was 44.12%). Neferine (10 μmol/L) caused a positive shift of the steady-state activation curve of Kv4.3 currents, and a negative shift of the steady-state inactivation curve. Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation. Neferine-induced blocking of Kv4.3 currents was frequency-dependent. In arterially perfused wedges of rabbit LV, neferine (1, 3, and 10 μmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, and caused dramatic increase of APD₁₀ at Epi sites.

Conclusion:

Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD₁₀ at Epi sites of rabbit heart.     

Effect of flecainide on action potentials and alternating current-induced arrhythmias in mammalian myocardium

The new antiarrhythmic drug flecainide (2,5-bis-(2,2,2-trifluoroethoxy)-N-(2-piperidylmethyl) benzamide acetate) increases action potential duration at 30 and 90% repolarization, and functional refractory period in guinea pig papillary muscle up to 10 mumol/L, but shortens the action potential and decreases its amplitude at 30 mumol/L, without significant change in resting potential. 10 mumol/L flecainide decreases maximal upstroke velocity (Vmax) by about 40% at a stimulation rate of 1 Hz, whereas at 0.017 Hz Vmax remains nearly unchanged (use-dependence). Flecainide delays recovery from inactivation of the fast-sodium channels. Its potential-dependent action on Vmax is demonstrated by a shift to more negative potentials of the membrane responsiveness curve and of the curve that relates membrane potential to Vmax in K+ depolarized papillary muscles driven at 0.017 Hz (h infinity -curve). Flecainide increases threshold of alternating current-induced arrhythmia and asystole in left atria and papillary muscles to a similar extent, and in this respect resembles the local anesthetics. Force of contraction of atrial and ventricular myocardium is significantly decreased at greater than or equal to 10 mumol/L flecainide, and frequency of spontaneously beating right atria at greater than or equal to 0.3 mumol/L. The results indicate that the predominant action of flecainide consists of a potent inhibition of cardiac fast sodium channels.     

Inhibition of Kv4.3 potassium channels by trazodone

Trazodone, a triazolopyridine antidepressant, is commonly used in the treatment of depression and insomnia. Kv4.3 channels are transiently, and rapidly, inactivating Kv channels that are highly expressed in cardiac myocytes and neurons. To determine the electrophysiological basis for the cardiac and neuronal actions of trazodone, we studied the effects of trazodone on Kv4.3 currents stably expressed in Chinese hamster ovary cells using the whole-cell patch-clamp technique. Trazodone decreased the peak amplitude of Kv4.3 in a concentration-dependent manner with an IC₅₀ of 55.4 μM. Under control conditions, the time course of inactivation of Kv4.3 at +40 mV was fitted to a double exponential function. Trazodone produced a concentration-dependent slowing of the fast and slow components of Kv4.3 inactivation during a voltage step to +40 mV. The inhibition of Kv4.3 by trazodone was voltage independent over the entire voltage range tested. Trazodone shifted the voltage dependence of the steady-state inactivation of Kv4.3 to a hyperpolarizing direction. However, the slope factor of the steady-state inactivation was not affected by trazodone. Under control conditions, the closed-state inactivation of Kv4.3 was fitted to a single exponential function. Trazodone significantly accelerated the closed-state inactivation of Kv4.3. Trazodone produced a weak use-dependent inhibition of Kv4.3 at frequencies of 1 and 2 Hz. m-Chlorophenylpiperazine (m-CPP), a major metabolite of trazodone, inhibited Kv4.3 less potently than trazodone, with an IC₅₀ of 118.6 μM. These results suggest that trazodone preferentially inhibited Kv4.3 by both binding to the closed state and accelerating the closed-state inactivation of the channel.     

Rosiglitazone inhibits Kv4.3 potassium channels by open-channel block and acceleration of closed-state inactivation

BACKGROUND AND PURPOSE

Rosiglitazone is a widely used oral hypoglycaemic agent, which improves insulin resistance in type 2 diabetes. Chronic rosiglitazone treatment is associated with a number of adverse cardiac events. The present study was designed to characterize the effects of rosiglitazone on cloned K_v4.3 potassium channels.

EXPERIMENTAL APPROACH

The interaction of rosiglitazone with cloned K_v4.3 channels stably expressed in Chinese hamster ovary cells was investigated using whole-cell patch-clamp techniques.

KEY RESULTS

Rosiglitazone decreased the currents carried by K_v4.3 channels and accelerated the current inactivation, concentration-dependently, with an IC₅₀ of 24.5 µM. The association and dissociation rate constants for rosiglitazone were 1.22 µM⁻¹·s⁻¹ and 31.30 s⁻¹ respectively. Block by rosiglitazone was voltage-dependent, increasing in the voltage range for channel activation; however, no voltage dependence was found in the voltage range required for full activation. Rosiglitazone had no effect on either the deactivation kinetics or the steady-state activation of K_v4.3 channels. Rosiglitazone shifted the steady-state inactivation curves in the hyperpolarizing direction, concentration-dependently. The K_i for the interaction between rosiglitazone and the inactivated state of K_v4.3 channels was 1.49 µM, from the concentration-dependent shift in the steady-state inactivation curves. Rosiglitazone also accelerated the kinetics of the closed-state inactivation of K_v4.3 channels. Rosiglitazone did not affect either use dependence or recovery from inactivation of K_v4.3 currents.

CONCLUSION AND IMPLICATIONS

Our results indicate that rosiglitazone potently inhibits currents carried by K_v4.3 channels by interacting with these channels in the open state and by accelerating the closed-state inactivation of K_v4.3 channels.

LINKED ARTICLE

This article is commented on by Hancox, pp. 496–498 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01281.x     

A single residue in the S6 transmembrane domain governs the differential flecainide sensitivity of voltage-gated potassium channels

Flecainide has been used to differentiate Kv4.2-based transient-outward K(+)-currents (flecainide-sensitive) from Kv1.4-based (flecainide-insensitive). We found that flecainide also inhibits ultrarapid delayed rectifier (I(Kur)) currents in Xenopus laevis oocytes carried by Kv3.1 subunits (IC(50), 28.3 +/- 1.3 microM) more strongly than Kv1.5 currents corresponding to human I(Kur) (IC(50), 237.1 +/- 6.2 microM). The present study examined molecular motifs underlying differential flecainide sensitivity. An initial chimeric approach pointed to a role for S6 and/or carboxyl-terminal sites in Kv3.1/Kv1.5 sensitivity differences. We then looked for homologous amino acid residues of the two sensitive subunits (Kv4.2 and Kv3.1) different from homologous residues for insensitive subunits (Kv1.4 and Kv1.5). Three candidate sites were identified: two in the S5-S6 linker and one in the S6 segment. Mutation of the proximal S5-S6 linker site failed to alter flecainide sensitivity. Mutation at the more distal site in Kv1.5 (V481L) modestly increased sensitivity, but the reciprocal Kv3.1 mutation (L401V) had no effect. S6 mutants caused marked changes: flecainide sensitivity decreased approximately 8-fold for Kv3.1 L422I (IC(50), 213 +/- 9 microM) and increased approximately 7-fold for Kv1.5 I502L (IC(50), 35.6 +/- 1.9 microM). Corresponding mutations reversed flecainide sensitivity of Kv1.4 and Kv4.2; L392I decreased Kv4.2 sensitivity by approximately 17-fold (IC(50) of 37.4 +/- 6.9 to 628 +/- 36 microM); I547L increased Kv1.4 sensitivity by approximately 15-fold (IC(50) of 706 +/- 37 to 40.9 +/- 7.3 microM). Our observations indicate that the flecainide sensitivity differences among these four voltage-gated K(+)-channels are determined by whether an isoleucine or a leucine is present at a specific amino acid location.     

Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis

1. In the present study, two new peptides, phrixotoxins PaTx1 and PaTx2 (29-31 amino acids), which potently block A-type potassium currents, have been purified from the venom of the tarantula Phrixotrichus auratus. 2. Phrixotoxins specifically block Kv4.3 and Kv4.2 currents that underlie I(to1), with an 5 < IC50 < 70 nM, by altering the gating properties of these channels. 3. Neither are the Shaker (Kv1), Shab (Kv2) and Shaw (Kv3) subfamilies of currents, nor HERG, KvLQT1/IsK, inhibited by phrixotoxins which appear specific of the Shal (Kv4) subfamily of currents and also block I(to1) in isolated murine cardiomyocytes. 4. In order to evaluate the physiological consequences of the Ito1 inhibition, mice were injected intravenously with PaTx1, which resulted in numerous transient cardiac adverse reactions including the occurrence of premature ventricular beats, ventricular tachycardia and different degrees of atrioventricular block. 5. The analysis of the mouse electrocardiogram showed a dose-dependent prolongation of the QT interval, chosen as a surrogate marker for their ventricular repolarization, from 249 +/- 11 to 265 +/- 8 ms (P < 0.05). 6. It was concluded that phrixotoxins, are new and specific blockers of Kv4.3 and Kv4.2 potassium currents, and hence of I(to1) that will enable further studies of Kv4.2 and Kv4.3 channel and/or I(to1) expression.     

Heteropoda toxin 2 interaction with Kv4.3 and Kv4.1 reveals differences in gating modification

Kv4 (Shal) potassium channels are responsible for the transient outward K(+) currents in mammalian hearts and central nervous systems. Heteropoda toxin 2 (HpTx2) is an inhibitor cysteine knot peptide toxin specific for Kv4 channels that inhibits gating of Kv4.3 in the voltage-dependent manner typical for this type of toxin. HpTx2 interacts with four independent binding sites containing two conserved hydrophobic amino acids in the S3b transmembrane segments of Kv4.3 and the closely related Kv4.1. Despite these similarities, HpTx2 interaction with Kv4.1 is considerably less voltage-dependent, has smaller shifts in the voltage-dependences of conductance and steady-state inactivation, and a 3-fold higher K(d) value. Swapping four nonconserved amino acids in S3b between the two channels exchanges the phenotypic response to HpTx2. To understand these differences in gating modification, we constructed Markov models of Kv4.3 and Kv4.1 activation gating in the presence of HpTx2. Both models feature a series of voltage-dependent steps leading to a final voltage-independent transition to the open state and closely replicate the experimental data. Interaction with HpTx2 increases the energy barrier for channel opening by slowing activation and accelerating deactivation. The greater degree of voltage-dependence in Kv4.3 occurs because it is the voltage-dependent transitions that are most affected by HpTx2; in contrast, it is the voltage-independent step in Kv4.1 that is most affected by the presence of toxin. These data demonstrate the basis for subtype-specificity of HpTx2 and point the way to a general model of gating modifier toxin interaction with voltage-gated ion channels.